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WO2020047002A4 - Method for transposase-mediated spatial tagging and analyzing genomic dna in a biological sample - Google Patents

Method for transposase-mediated spatial tagging and analyzing genomic dna in a biological sample Download PDF

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WO2020047002A4
WO2020047002A4 PCT/US2019/048425 US2019048425W WO2020047002A4 WO 2020047002 A4 WO2020047002 A4 WO 2020047002A4 US 2019048425 W US2019048425 W US 2019048425W WO 2020047002 A4 WO2020047002 A4 WO 2020047002A4
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Prior art keywords
sequence
biological sample
capture
enzyme
genomic dna
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WO2020047002A1 (en
Inventor
Michael Schnall-Levin
Michael Ybarra Lucero
Tarjei Sigurd Mikkelsen
Patrik Stahl
Jonas Frisen
Maja MARKLUND
Enric LLORENS
Eswar Prasad Ramachandran Iyer
Lucas Frenz
Augusto Manuel TENTORI
Rajiv Bharadwaj
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10X Genomics Inc
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10X Genomics Inc
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Priority to CN201980071274.0A priority Critical patent/CN113366117B/en
Priority to CN202511001938.XA priority patent/CN120775968A/en
Priority to US18/023,257 priority patent/US20230323447A1/en
Priority to EP19773221.7A priority patent/EP3844307A1/en
Priority to SG11202102019UA priority patent/SG11202102019UA/en
Publication of WO2020047002A1 publication Critical patent/WO2020047002A1/en
Publication of WO2020047002A4 publication Critical patent/WO2020047002A4/en
Priority to US16/876,709 priority patent/US20210010070A1/en
Priority to US16/876,682 priority patent/US20200407781A1/en
Anticipated expiration legal-status Critical
Priority to US17/688,241 priority patent/US11519033B2/en
Priority to US17/933,347 priority patent/US20230340596A1/en
Priority to US18/348,072 priority patent/US12270077B2/en
Priority to US18/410,474 priority patent/US20240191286A1/en
Priority to US18/987,997 priority patent/US12378607B2/en
Priority to US18/988,119 priority patent/US12344892B2/en
Priority to US19/020,320 priority patent/US20250146071A1/en
Priority to US19/020,508 priority patent/US20250146072A1/en
Priority to US19/020,235 priority patent/US20250146057A1/en
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation

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  • General Health & Medical Sciences (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measurement Of The Respiration, Hearing Ability, Form, And Blood Characteristics Of Living Organisms (AREA)
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Abstract

The present disclosure relates to materials and methods for spatially analyzing nucleic acids that have been fragmented with a transposase enzyme, alone or in combination with other types of analytes

Claims

AMENDED CLAIMS received by the International Bureau on 16 March 2020 (16.03.2020)
1. A method for spatial analysis of genomic DNA present in a biological sample, the method comprising:
providing an array comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises a spatial barcode and a capture domain;
permeabilizing the biological sample under conditions sufficient to make the genomic DNA in the biological sample accessible to transposon insertion;
providing a transposon sequence and a transposase enzyme to the biological sample under conditions wherein the transposon sequence is inserted into the genomic DNA;
allowing the transposase enzyme to excise the inserted transposon sequence from the genomic DNA, thereby generating fragmented genomic DNA;
contacting the biological sample comprising the fragmented genomic DNA with the array under conditions where the capture probe interacts with the fragmented genomic DNA; and correlating the location of the capture probe on the array to a location in the biological sample, thereby spatially analyzing the fragmented genomic DNA.
2. The method of claim 1, wherein the array comprising the plurality of capture probes is provided on a substrate.
3. The method of claim 1, wherein the array comprising the plurality of capture probes is provided on a feature.
4. The method of any one of claims 1 to 3, wherein the capture probe is directly or
indirectly attached.
5. The method of any one of claims 1 to 4, wherein the array comprising the plurality of capture probes is provided on the feature on the substrate.
6. The method of any one of claims 1 to 5, wherein the substrate comprises a microfluidic channel.
7. The method of any one of claims 1 to 6, wherein the capture probe further comprises one or more of a cleavage domain, a functional domain, and a unique identifier, or combinations thereof.
8. The method of any one of claims 1 to 7, further comprising a migration step wherein the fragmented genomic DNA is migrated to the substrate.
9. The method of claim 8, wherein the migration step is an active migration step comprising applying an electric field to the fragmented genomic DNA.
10. The method of claim 8, wherein the migration step is a passive migration step comprising diffusion.
11. The method of any one of claims 8 to 10, wherein the migration of the fragmented genomic DNA from the biological sample comprises exposing the biological sample and the feature to heat.
12. The method of any one of claims 1 to 11, wherein the biological sample is immobilized on the substrate.
13. The method of any one of claims 1 to 12, wherein the transposase enzyme is a dimer comprised of a first monomer complexed with a first adapter comprising a transposon end sequence and a sequence complementary to the capture domain and wherein a second monomer is complexed with a second adapter comprising a transposon end sequence and a second adapter sequence, wherein the transposase enzyme ligates the first adapter and the second adapter to the fragmented genomic DNA.
14. The method of claim 13, wherein the first adapter and the second adapter have a 5’ end and a 3’ end, wherein the 5’ end is phosphorylated in situ.
15. The method of claim 13 or 14, wherein prior to fragmenting the DNA, the 5’ end of the first adapter complexed with the first monomer and the second adapter complexed with the second monomer are phosphorylated.
16. The method of claim 14 or 15, wherein the step of phosphorylating the 5’ end of the first adapter complexed with the first monomer and the second adapter complexed with the second monomer comprises contacting a first monomer: first adapter complex and a second monomer: second adapter complex with a polynucleotide kinase in the presence of ATP.
17. The method of any one of claims 1 to 16, wherein the capture domain of the capture probe comprises a sequence that hybridizes to the sequence complementary to the capture domain of the first adapter.
18. The method of any one of claims 13 to 17, wherein the capture probe is a partially double stranded molecule comprising a first strand comprising the capture domain hybridized to a second strand, and wherein the first strand templates the ligation of the first adapter to the second strand.
339
19. The method of any one of claims 13 to 18, wherein the first adapter sequence
complementary to the capture domain, or portion thereof, hybridized to the capture probe templates the ligation and ligating the 5’ end of the first adapter to the 3’ end of the capture probe.
20. The method of any one of claims 1 to 19, wherein the capture probe comprises a surface probe and a splint oligonucleotide and wherein the splint oligonucleotide comprises a sequence complementary to a hybridization domain of the surface probe, or portion thereof.
21. The method of claim 20, wherein the splint oligonucleotide comprises the capture domain with a sequence complementary to the first adapter, or portion thereof.
22. The method of claim 20 or 21, wherein the splint oligonucleotide hybridizes to the first adapter, or portion thereof, and to the hybridization domain of the surface probe, or portion thereof.
23. The method of any one of claims 20 to 22, wherein ligation is performed in the presence of the splint oligonucleotide, thereby ligating the surface probe of the capture probe and the first adapter.
24. The method of any one of claims 13 to 23, wherein the fragmented genomic DNA
hybridized to the capture probe by the first adapter is an extension template used to
340 produce an extended capture probe that comprises the sequences of the spatial barcode and a sequence complementary to the fragmented genomic DNA.
25. The method of claim 24, wherein the capture probe hybridized to the fragmented
genomic DNA is extended with a DNA polymerase.
26. The method of claim 25, wherein the DNA polymerase has strand displacement activity.
27. The method of any one of claims 1 to 26, further comprising gap repair of single stranded breaks in the fragmented genomic DNA.
28. The method of any one of claims 13 to 26, wherein the sequence complementary to the capture domain is a unique sequence.
29. The method of any one of claims 1-28, wherein the capture probe is ligated to the
fragmented genomic DNA by a DNA ligase enzyme.
30. The method of any one of claims 1 to 29, wherein the transposase enzyme is a Tn5
transposase enzyme, or a functional derivative thereof.
31. The method of claim 30, wherein the Tn5 transposase enzyme comprises a sequence having at least 80% identity to SEQ ID NO: 1.
32. The method of any one of claims 1 to 29, wherein the transposase enzyme is a Mu
transposase enzyme, or the functional derivative thereof.
341
33. The method of claim 32, wherein the Mu transposase enzyme comprises a sequence having at least 80% identity to SEQ ID NO: 2.
34. The method of claim 13, wherein the transposon end sequence comprises a sequence having at least 80% identity to SEQ ID NO: 8.
35. The method of claim 13, wherein the transposon end sequence comprises a sequence having at least 80% identity to any one of SEQ ID NO: 9 to 14.
36. The method of any one of claims 1 to 35, wherein permeabilizing the biological sample is performed under a chemical permeabilization condition, an enzymatic permeabilization condition, or both.
37. The method of claim 36, wherein the chemical permeabilization condition comprises contacting the biological sample with an alkaline solution.
38. The method of claim 36 or 37, wherein the enzymatic permeabilization condition
comprises contacting the biological sample with an acidic solution comprising a protease enzyme.
39. The method of claim 38, wherein the protease enzyme is an aspartyl protease, preferably a pepsin enzyme, a pepsin-like enzyme, or the functional equivalent thereof.
342
40. The method of claim 39, wherein the pepsin enzyme, the pepsin-like enzyme, or the functional equivalent thereof, comprises a sequence having at least 80% identity to SEQ ID NO: 3 or 4.
41. The method of any one of claims 36-40, wherein the enzymatic permeabilization condition comprises contacting the biological sample with:
(i) a zinc endopeptidase, a collagenase enzyme, a collagenase-like enzyme, or a functional equivalent thereof;
(ii) a serine protease, a proteinase K enzyme, a proteinase K-like enzyme, or a functional equivalent thereof; or
(iii) both.
42. The method of claim 41, wherein the collagenase enzyme, the collagenase-like enzyme, or the functional equivalent thereof comprises a sequence having at least 80% identity to SEQ ID NO: 5 or 6.
43. The method of claim 41 or 42, wherein the proteinase K enzyme, the proteinase K-like enzyme, or the functional equivalent thereof comprises a sequence having at least 80% identity to SEQ ID NO: 7.
44. The method of claim 25 or 26, wherein using the fragmented genomic DNA hybridized to the capture probe as the extension template generates a DNA molecule.
45. The method of claim 18 or 19, where using the fragmented genomic DNA hybridized to the capture probe as a ligation template generates a DNA molecule.
46. The method of claim 44 or 45, comprising a step of analyzing the DNA molecule.
343
47. The method of claim 46, wherein the step of analyzing the DNA molecule includes sequencing.
48. The method of claim any one of claims 1 to 47, wherein correlating the spatial barcode of the capture probe with the fragmented genomic DNA associated with the capture probe spatially analyzes the fragmented genomic DNA.
49. The method of any one of claims 1 to 48, further comprising a step wherein the biological sample is imaged before or after contacting the biological sample with the substrate.
50. A kit for use in a method of spatially detecting nucleic acids of a biological sample as defined in any one of claims 1 to 49, wherein the kit comprises any two or more of:
(i) an array on which a plurality of capture probes are present;
(ii) one or more biological sample permeabilization reagents;
(iii) one or more transposase enzymes;
(iv) one or more reverse transcriptases; and
(v) one or more cleavage enzymes.
51. A method for spatial analysis of genomic DNA and RNA present in a biological sample, the method comprising:
providing an array, wherein the array comprises a plurality of capture probes, wherein a first capture probe of the plurality of capture probes comprises a spatial barcode and a first capture domain, and wherein a second capture probe of the plurality of capture probes comprises the spatial barcode and a second capture domain;
permeabilizing the biological sample under conditions sufficient to make the genomic DNA in the biological sample accessible to transposon insertion;
providing a transposon sequence and a transposase enzyme to the biological sample under conditions wherein the transposon sequence is inserted into the genomic DNA;
344 allowing the transposase enzyme to excise the inserted transposon sequence from the genomic DNA, thereby generating fragmented genomic DNA;
contacting the biological sample comprising the fragmented genomic DNA and RNA with the array under conditions where the first capture domain interacts with the fragmented genomic DNA and the second capture domain interacts with the RNA; and
correlating the location of the first capture probe on the array to a location in the biological sample and correlating the location of the second capture probe on the array to a location in the biological sample, thereby spatially analyzing the fragmented genomic DNA and RNA at the location in the biological sample.
52. The method of claim 51 , wherein the RNA is a mRNA.
53. The method of claim 51 or 52, wherein the first capture domain and the second capture domain are identical.
54. The method of claim 53, wherein the first capture domain and the second capture domain comprise a homopolymeric poly(T) sequence.
55. The method of any one of claims 51 to 54, wherein the first capture domain and the second capture domain are different.
56. The method of claim 55, wherein the first capture domain comprises a random sequence and the second capture domain comprises a poly(T) sequence.
57. The method of any one of claims 51 to 56, wherein the array comprising the plurality of capture probes is provided on a substrate.
58. The method of any one of claims 51 to 56, wherein the array comprising the plurality of capture probes is provided on a feature.
345
59. The method of claim 58, wherein the feature comprises the first capture probe, the second capture probe, or both.
60. The method of any one of claims 51 to 59, wherein the first capture probe, the second capture probe, or both, are directly or indirectly attached.
61. The method of any one of claims 51 to 60, wherein the array comprising the plurality of capture probes is provided on the feature on the substrate.
62. The method of any one of claims 51 to 61, wherein the substrate comprises a microfluidic channel.
63. The method of any one of claims 51 to 62, wherein the first capture probe, the second capture probe, or both, comprise one or more of a cleavage domain, a functional domain, and a unique identifier, or combinations thereof.
64. The method of any one of claims 51 to 63, comprising a migration step wherein the fragmented genomic DNA and the RNA are migrated to the substrate.
65. The method of claim 64, wherein the migration step is an active migration step.
66. The method of claim 64, wherein the migration step is a passive migration step.
67. The method of any one of claims 64 to 66, wherein the migration of the fragmented genomic DNA and the RNA from the biological sample comprises exposing the biological sample to heat.
68. The method of any one of claims 51 to 67, wherein the biological sample is immobilized on the substrate.
346
69. The method of any one of claims 51 to 68, wherein the fragmented genomic DNA is repaired by ligating breaks with a ligase enzyme.
70. The method of any one of claims 51 to 69, further comprising gap repair of single stranded breaks in the fragmented genomic DNA.
71. The method of any one of claims 51 to 70, wherein a sequence complementary to the first capture domain of the first capture probe is introduced to the fragmented genomic DNA.
72. The method of any one of claims 51 to 71, wherein the first capture domain of the first capture probe hybridizes to the sequence complementary to the capture domain introduced to the fragmented genomic DNA.
73. The method of claim 57, wherein the random sequence of the first capture domain hybridizes the fragmented genomic DNA.
74. The method of any one of claims 51 to 73, wherein the second capture domain of the second capture probe hybridizes to a complementary sequence in the mRNA.
75. The method of any one of claims 71 or 74, wherein the sequence complementary to the first capture domain and the complementary sequence in the mRNA is a homopolymeric sequence.
76. The method of claim 75, wherein the homopolymeric sequence is a poly(A) sequence.
77. The method of any one of claims 51-76, comprising extending the first capture probe using the fragmented genomic DNA as an extension template, and extending the second capture probe using the RNA as an extension template.
347
78. The method of claim 77, wherein extending the first capture probe is performed with a DNA polymerase.
79. The method of claim 78, wherein extending the second capture probe is performed with reverse transcriptase.
80. The method of any one of claims 51 to 79, wherein the transposase enzyme is a Tn5 transposase enzyme, or a functional derivative thereof.
81. The method of claim 80, wherein the Tn5 transposase enzyme comprises a sequence having at least 80% identity to SEQ ID NO: 1.
82. The method of any one of claims 51 to 79, wherein the transposase enzyme is a Mu transposase enzyme, or a functional derivative thereof.
83. The method of claim 82, wherein the Mu transposase comprises a sequence having at least 80% identity to SEQ ID NO: 2.
84. The method of claim any one of claims 51 to 83, wherein the transposase enzyme is complexed with an adapter comprising a transposon end sequence.
85. The method claim 84, wherein the transposon end sequence comprises a sequence having at least 80% identity to SEQ ID NO: 8.
86. The method of claim 84, wherein the transposon end sequence comprises a sequence having at least 80% identity to any one of SEQ ID NO: 9 to 14.
87. The method of any one of claims 51 to 86, wherein permeabilizing the biological sample is performed under a chemical permeabilization condition, an enzymatic
permeabilization condition, or both.
348
88. The method of claim 87, wherein the chemical permeabilization condition comprises contacting the biological sample with an alkaline solution.
89. The method of claim 86 or 87, wherein the enzymatic permeabilization condition comprises contacting the biological sample with an acidic solution comprising a protease enzyme.
90. The method of claim 89, wherein the protease enzyme is an aspartyl protease, preferably a pepsin enzyme, a pepsin-like enzyme, or a functional equivalent thereof.
91. The method of claim 90, wherein the pepsin enzyme, the pepsin-like enzyme, or functional equivalent thereof, comprises a sequence having at least 80% identity to SEQ ID NO: 3 or 4.
92. The method of any one of claims 86 to 91, wherein the enzymatic permeabilization condition comprises contacting the biological sample with:
(i) a zinc endopeptidase, a collagenase enzyme, a collagenase-like enzyme, or a functional equivalent thereof;
(ii) a serine protease, a proteinase K enzyme, a proteinase K-like enzyme, or a functional equivalent thereof; or
(iii) both.
93. The method of claim 92, wherein the collagenase enzyme, the collagenase-like enzyme, or the functional equivalent thereof comprises a sequence having at least 80% identity to SEQ ID NO: 5 or 6.
94. The method of claim 92 or 93, wherein the proteinase K enzyme, the proteinase K- like enzyme, or the functional equivalent thereof comprises a sequence having at least 80% identity to SEQ ID NO: 7.
349
95. The method of claim any one claims 51 to 94, wherein the step of analyzing the DNA molecule includes sequencing.
96. The method of any one of claims 51 to 95, wherein correlating the spatial barcode of the first capture probe with the fragmented genomic DNA associated with the first capture probe spatially analyzes the fragmented genomic DNA.
97. The method of any one of claims 51 to 96, wherein correlating the spatial barcode of the second capture probe with the mRNA associated with the second capture probe spatially analyzes the mRNA.
98. The method of any one of claims 51 to 97, further comprising a step wherein the biological sample is imaged before or after contacting the biological sample with the substrate.
99. A method for determining a location of an analyte in a biological sample, comprising:
(a) contacting a biological sample with a substrate comprising a plurality of capture probes, wherein a capture probe of the plurality comprises a spatial barcode and a capture domain that specifically binds to an analyte from the biological sample; and
(b) determining (i) all or a portion of the analyte sequence specifically bound to the capture domain, or a complement thereof, and (ii) all or a portion of the spatial barcode, or a complement thereof, and using the determined sequences of (i) or (ii) to determine the location of the analyte in the biological sample, wherein the biological sample is a cell culture, an organism, or an organoid.
100. The method of claim 99, wherein the biological sample is permeabilized prior to step (b).
101. The method of claim 99, further comprising permeabilizing the biological sample prior to step (b).
350
102. The method of claim 101, further comprising fixing the biological sample, prior to the step of permeabilizing the biological sample.
103. The method of any one of claims 99-102, wherein the organism is a nematode, a plant, a fungi, an insect, an arachnid, a fish, an amphibian, or a reptile.
104. The method of any one of claims 99- 102, wherein the cell culture comprises a plurality of cells.
105. The method of claim 104, wherein the plurality of cells are non-adherent cells.
106. The method of claim 104 or 105, wherein the plurality of cells is from disassociated tissue or a tissue section.
107. The method of any one of claims 104- 106, wherein a cell of the plurality of cells is immobilized on the substrate after the cell is distributed onto the substrate.
108. The method of claim 107, wherein the cell of the plurality of cells is immobilized on the substrate by a polymer coating.
109. The method of claim 107, wherein the cell of the plurality of cells is immobilized on the substrate by a lectin, poly-lysine, an antibody, or a polysaccharide.
110. The method of claim 104, wherein the plurality of cells are adherent cells.
111. The method of claim 110, wherein the adherent cells are from a cell line selected from the group consisting of: BT549, HS 578T, MCF7, MDA-MB-231, MDAMB-468, T-47D, SF268, SF295, SF539, SNB-19, SNB-75, U251, Colo205, HCC 2998, HCT-116, HCT-15,
HT29, KM12, SW620, 786-0, A498, ACHN, CAKI, RXF 393, SN12C, TK-10, UO-31, A549,
351 EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H460, NCI-H522, LOX IMVI, M14, MALME-3M, MDA-MB-435, SK-, EL-2, SK-MEL-28, SK-MEL-5, UACC-257, UACC-62, IGROV1, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, SK-OV-3, NCI-ADR-RES, DU145, PC-3, DU145, H295R, HeLa, KBM-7, LNCaP, MCF-7, MDA-MB-468, PC3, SaOS-2, SH- SY5Y, T-47D, THP-l, U87, vero, MC3T3, GH3, PC12, dog MDCK kidney epithelial, Xenopus A6 kidney epithelial, zebrafish AB9, and Sf9 insect epithelial cell lines.
112. The method of any one of claims 99-111, further comprising imaging the biological sample.
113. The method of claim 112, wherein the imaging is used to determine a region of interest in the biological sample.
114. The method of claim 112, wherein the imaging is used to determine morphology of the biological sample or a region of the biological sample.
115. The method of claim 114, further comprising correlating the morphology of the biological sample or the region of the biological sample to the location of the analyte in the biological sample.
116. The method of any one of claims 99-115, wherein the analyte is DNA.
117. The method of any one of claims 99-115, wherein the analyte is RNA.
118. The method of claim 117, wherein the capture domain comprises a poly-dT sequence.
119. The method of any one of claims 99-118, wherein the capture probe further comprises a unique molecular identifier (UMI).
352
120. The method of any one of claims 99-119, wherein the capture probe further comprises a functional domain.
121. The method of any one of claims 99-120, wherein the capture probe further comprises a cleavage domain.
122. A method for spatial analysis of a nucleic acid in a biological sample, the method comprising:
(a) contacting the biological sample to an array comprising a plurality of capture probes immobilized on the array, wherein a capture probe of the plurality of capture probes comprises a spatial barcode and a capture domain;
(b) contacting a polymer material to the biological sample, and activating the polymer material to form a hydrogel-embedded biological sample;
(c) permeabilizing the hydrogel-embedded biological sample under conditions
sufficient to allow the nucleic acid in the biological sample to specifically bind to the capture domain;
(d) amplifying the RNA specifically bound to the capture domain to generate an
amplicon having a sequence, wherein the amplicon comprises all or a portion of the spatial barcode or a complement thereof, and all or a portion of the nucleic acid sequence or a complement thereof; and
(e) determining the sequence of the amplicon by sequential hybridization and detection with a plurality of labelled probes, and using the determined amplicon sequence to identify the location of the nucleic acid in the biological sample.
123. The method of claim 122, wherein step (d) comprises amplifying the nucleic acid specifically bound to the capture domain in situ.
124. The method of claim 122 or 123, wherein step (e) comprises sequencing the amplicon.
353
125. The method of any one of claims 122-124, wherein the plurality of labelled probes is a plurality of fluorescently- labelled probes.
126. The method of any one of claims 122-125, wherein the permeabilizing in step (c) is performed using chemical permeabilization conditions, enzymatic permeabilization conditions, or both.
127. The method of any one of claims 122-126, wherein the biological sample comprises a tissue section.
128. The method of any one of claims 122-127, wherein the nucleic acid is RNA
129. The method of any one of claims 122-127, wherein the nucleic acid is DNA.
130. A nucleic acid comprising:
(a) a first single-stranded oligonucleotide comprising:
a spatial barcode;
a primer binding site;
a unique molecular identifier; and
a first hybridization sequence; and
(b) a splint oligonucleotide comprising:
a second hybridization sequence that specifically binds to the first hybridization sequence; and
a capture domain.
131. The nucleic acid of claim 130, wherein the first single-stranded oligonucleotide comprises, in a 5’ to a 3’ direction:
the spatial barcode;
the primer binding site;
the unique molecular identifier; and
354 the first hybridization sequence.
132. The nucleic acid of claim 131, wherein a 5’ end of the first single-stranded oligonucleotide is attached to a substrate.
133. The nucleic acid of any one of claims 130-132, wherein the splint oligonucleotide comprises, in a 5’ to 3’ direction the capture domain and the second hybridization sequence.
134. The nucleic acid of claim 133, wherein the capture domain comprises a sequence that is capable of specifically binding to a sequence present in a single-stranded tagment.
135. The nucleic acid of claim 134, wherein the sequence present in the single-stranded tagment is a sequence present in a first adapter.
136. A composition comprising:
(a) a nucleic acid of any one of claims 130-133; and
(b) a single-stranded tagment comprising a first adaptor that comprises a sequence that is capable of specifically binding to the capture domain.
137. The composition of claim 136, wherein the single-stranded tagment comprises the first adaptor sequence at its 5’ end.
138. The composition of claim 136 or 137, wherein the first adaptor sequence further comprises a first adaptor transposon end sequence.
139. The composition of claim 138, wherein the first adaptor sequence comprises, in a 5’ to 3’ direction, the sequence that is capable of specifically binding to the capture domain and the first adaptor transposon end sequence.
140. The composition of any one of claims 136-139, wherein the single-stranded tagment further comprises a second adaptor.
355
141. The method of any one of claims 136-138, wherein the single-stranded tagment comprises the second adaptor at its 3’ end.
142. The method of claim 141, wherein the second adaptor sequence comprises a second adaptor transposon end sequence and a primer binding site or a complement thereof.
143. A composition comprising:
(a) a nucleic acid of any one of claims 130-133;
(b) DNA from a biological sample; and
(c) a transposase dimer comprising:
(i) a first transposase monomer complexed with a first adaptor comprising a first transposon end sequence and a sequence that is capable of specifically binding to the capture domain;
(ii) a second transposase monomer complexed with a second adaptor comprising a second transposon end sequence and a primer binding site or a complement thereof.
144. The composition of claim 143, wherein a 5’ end of the first adaptor is
phosphorylated.
145. The composition of claim 143, wherein a 5’ end of the second adaptor is phosphorylated.
146. A composition comprising:
(a) a nucleic acid of any one of claims 130-133;
(b) DNA from a biological sample; and
(c) a first transposase monomer complexed with a first adaptor comprising a first transposon end sequence and a sequence that is capable of specifically binding to the capture domain; and
356 (d) a second transposase monomer complexed with a second adaptor comprising a second transposon end sequence and a primer binding site or a complement thereof.
147. The composition of claim 146, wherein a 5’ end of the first adaptor is phosphorylated.
148. The composition of claim 146, wherein a 5’ end of the second adaptor is phosphorylated.
357
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US18/023,257 US20230323447A1 (en) 2018-08-28 2019-08-27 Method for transposase-mediated spatial tagging and analyzing genomic dna in a biological sample
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US16/876,709 US20210010070A1 (en) 2018-08-28 2020-05-18 Method for transposase-mediated spatial tagging and analyzing genomic dna in a biological sample
US16/876,682 US20200407781A1 (en) 2018-08-28 2020-05-18 Method for transposase-mediated spatial tagging and analyzing genomic dna in a biological sample
US17/688,241 US11519033B2 (en) 2018-08-28 2022-03-07 Method for transposase-mediated spatial tagging and analyzing genomic DNA in a biological sample
US17/933,347 US20230340596A1 (en) 2018-08-28 2022-09-19 Method for transposase-mediated spatial tagging and analyzing genomic dna in a biological sample
US18/348,072 US12270077B2 (en) 2018-08-28 2023-07-06 Method for transposase-mediated spatial tagging and analyzing genomic DNA in a biological sample
US18/410,474 US20240191286A1 (en) 2018-08-28 2024-01-11 Method for transposase-mediated spatial tagging and analyzing genomic dna in a biological sample
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